Modulation of inflammation by hops fractions and derivatives

ABSTRACT

A natural formulation of compounds that would to modulate inflammation is disclosed. The formulation would also inhibit expression of COX-2, inhibit synthesis of prostaglandins selectively in target cells, and inhibit inflammatory response selectively in target cells. The compositions containing at least one fraction isolated or derived from hops.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application is a continuation-in-part of U.S.application Ser. No. 10/400,293, filed Mar. 26, 2003, and acontinuation-in-part of U.S. application Ser. No. 10/401,283, filed Mar.26, 2003, both of which claim the benefit under 35 U.S.C. § 119(e) toprovisional application No. 60/450,237, filed on Feb. 25, 2003, andprovisional application No. 60/420,383, filed on Oct. 21, 2002. Thisapplication is also a continuation-in-part of U.S. application Ser. No.09/885,721, filed Jun. 20, 2001. The contents of each of these earlierapplications are hereby incorporated by reference as if recited hereinin their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to methods of use of acomposition comprising hops extracts or derivatives thereof, wherein thecompositions can inhibit expression of cyclooxygenase-2 (COX-2), inhibitsynthesis of prostaglandins selectively in target cells, and/or inhibitinflammatory response selectively in target cells.

[0004] 2. Description of the Related Art

[0005] Cyclooxygenase (prostaglandin endoperoxide synthase, EC 1.14.991,COX) catalyzes the rate-limiting step in the metabolism of arachidonicacid to prostaglandin H₂ (PGH₂), which is further metabolized to variousprostaglandins, prostacyclin and thromboxane A2 (c.f. FIG. 1). In theearly 1990s, it was established that COX exists in two isoforms,commonly referred to as COX-1 and COX-2. It was subsequently determinedthat the COX-1 and COX-2 proteins are derived from distinct genes thatdiverged well before birds and mammals. Prostaglandins (PGs) generatedvia the COX-1 and COX-2 pathways are identical molecules and thereforehave identical biological effects. COX-1 and COX-2, however, maygenerate a unique pattern and variable amounts of eicosanoids;therefore, relative differences in the activation of these isozymes mayresult in quite dissimilar biological responses. Differences in thetissue distribution and regulation of COX-1 and COX-2 are now consideredcrucial for the beneficial as well as adverse effects of COX inhibitors.

[0006] The generally held concept (COX dogma) is that COX-1 is expressedconstitutively in most tissues whereas COX-2 is the inducible enzymetriggered by pro-inflammatory stimuli including mitogens, cytokines andbacterial lipopolysaccharide (LPS) in cells in vitro and in inflamedsites in vivo. Based primarily on such differences in expression, COX-1has been characterized as a housekeeping enzyme and is thought to beinvolved in maintaining physiological functions such as cytoprotectionof the gastric mucosa, regulation of renal blood flow, and control ofplatelet aggregation. COX-2 is considered to mainly mediateinflammation, although constitutive expression is found in brain, kidneyand the gastrointestinal tract. Therefore, it would be desirable todown-regulate tissue-specific or cell-specific expression of COX-2.

[0007] Arachidonic acid serves as the primary substrate for thebiosynthesis of all PGs. PGs are ubiquitous hormones that function asboth paracrine and autocrine mediators to affect a myriad ofphysiological changes in the immediate cellular environment. The variedphysiological effects of PGs include inflammatory reactions such asrheumatoid arthritis and osteoarthritis, blood pressure control,platelet aggregation, induction of labor and aggravation of pain andfever. The discovery 30 years ago that aspirin and other non-steroidalanalgesics inhibited PG production identified PG synthesis as a targetfor drug development. There are at least 16 different PGs in ninedifferent chemical classes, designated PGA to PGI. PGs are part of alarger family of 20-carbon-containing compounds called eicosanoids; theyinclude prostacyclins, thromboxanes, and leukotrienes. The array of PGsproduced varies depending on the downstream enzymatic machinery presentin a particular cell type. For example, endothelial cells produceprimarily PGI₂, whereas platelets mainly produce TXA₂.

[0008] Prostaglandins (PG) are believed to play an important role inmaintenance of human gastric mucosal homeostasis. Current dogma is thatCOX-1 is responsible for PG synthesis in normal gastric mucosa in orderto maintain mucosal homeostasis and that COX-2 is expressed by normalgastric mucosa at low levels, with induction of expression during ulcerhealing, following endotoxin exposure or cytokine stimulation. It nowappears that both COX-1 and COX-2 have important physiological roles inthe normal gastric mucosa.

[0009] Compounds that inhibit the production of PGs by COX have becomeimportant drugs in the control of pain and inflammation. Collectivelythese agents are known as non-steroidal anti-inflammatory drugs (NSAIDs)with their main indications being osteoarthritis and rheumatoidarthritis. However, the use of NSAIDs, and in particular aspirin, hasbeen extended to prophylaxis of cardiovascular disease. Over the lastdecade, considerable effort has been devoted to developing new moleculesthat are direct inhibitors of the enzymatic activity of COX-2, with theinference that these compounds would be less irritating to the stomachwith chronic use. Therefore, it would be desirable to inhibitinflammation response selectively in target cells.

[0010] U.S. patent application 2002/0086070A1 of Kuhrts entitled,“ANTI-INFLAMMATORY AND CONNECTIVE TISSUE REPAIR FORMULATIONS” describesa hops component that has an IC₅₀-WHMA COX-2/COX-1 ratio ranging fromabout 0.23 to about 3.33. Example 1 of the application describes acomposition containing an extract obtained through supercritical carbondioxide extraction of whole hops (CO₂-extract) comprising 42% humulone.

[0011] U.S. Pat. No. 6,391,346 entitled, “ANTI-INFLAMMATORY,SLEEP-PROMOTING HERBAL COMPOSITION AND METHOD OF USE” describes anorally administered composition capable of reducing inflammation inanimals, while promoting sleep for such animals. The compositioncontains hydroalcoholic extract of hops and supercritical carbon dioxideextract of hops which are used to promote sleep.

[0012] An ideal formulation for the treatment of inflammation wouldinhibit the induction and activity of COX-2 without inhibiting thesynthesis of PGE₂ in gastric mucosal cells. However, conventionalnon-steroidal anti-inflammatory drugs lack the specificity of inhibitingCOX-2 without affecting gastric PGE₂ synthesis and are at risk to causedamages on the gastrointestinal system, when used for extended periods.Indeed, even the newly developed, anti-inflammatory drugs such asrofecoxib and celexocib produce untoward gastric toxicity in the form ofinduced spontaneous bleeding and delay of gastric ulcer healing.

[0013] Thus, it would be useful to identify a formulation of compoundsthat would specifically inhibit or prevent the synthesis ofprostaglandins by COX-2 with little or no effect on synthesis of PGE₂ inthe gastric mucosa. Such a formulation, which would be useful forpreserving the health of joint tissues, for treating arthritis or otherinflammatory conditions, has not previously been discovered. The term“specific or selective COX-2 inhibitor” was coined to embrace compoundsor mixtures of compounds that selectively inhibit COX-2 over COX-1.However, while the implication is that such a calculated selectivitywill result in lower gastric irritancy, unless the test materials areevaluated in gastric cells, the term “selective COX-2 inhibitor” doesnot carry assurance of safety to gastrointestinal cells. Only testing ofcompound action in target tissues, inflammatory cells and gastricmucosal cells, will identify those agents with low potential for stomachirritation.

[0014] The major problem associated with ascertaining COX-2 selectivity(i.e. low gastric irritancy) is that differences in assay methodologycan have profound effects on the results obtained. Depicted in Table 1are the categories of the numerous in vitro assays that have beendeveloped for testing and comparing the relative inhibitory activitiesof NSAID and natural compounds against COX-1 and COX-2. These testsystems can be classified into three groups: (1) systems using animalenzymes, animal cells or cell lines, (2) assays using human cell lines,or human platelets and monocytes, and (3) currently evolving modelsusing human cells that are representative of the target cells for theanti-inflammatory and adverse effects of NSAID and dietary supplements.Generally, models using human cell lines or human platelets andmonocytes are the current standard and validated target cell models havenot been forthcoming. A human gastric cell line capable of assessingpotential for gastric irritancy is a need. TABLE 1 Classification oftest systems for in vitro assays assessing COX-2 selectivity ofanti-inflammatory compounds† TEST SYSTEMS ANIMAL HUMAN TARGET EnzymesEnzymes Human Gastric Mucosa Cells Cells Cells Human Chondrocytes Celllines Cell lines Human Synoviocytes OTHER SYSTEM VARIABLES 1. Source ofarachidonic acid-endogenous or exogenous; 2. Various expression systemsfor gene replication of COX-1 and COX-2; 3. The presence or absence of aCOX-2 inducing agent; 4. COX-2 inducing agents are administered atdifferent concentrations and for different periods of time; 5. Durationof incubation with the drug or with arachidonic acid; 6. Variation inthe protein concentration in the medium.

[0015] The enzymes used can be of animal or human origin, they can benative or recombinant, and they can be used either as purified enzymes,in microsomal preparations, or in whole-cell assays. Other systemvariables include the source of arachidonic acid. PG synthesis can bemeasured from endogenously released arachidonc acid or exogenously addedarachidonic acid. In the later case, different concentrations are usedin different laboratories.

[0016] Second, there are various expression systems for gene replicationof recombinant COX-1 and COX-2 enzymes. In addition, the cellstransfected with the Cox-1 or Cox-2 gene can be of diverse origins, forinstance, insect cell lines or COS cells. Third, the absence or presenceof a COX-2 inducing agent can vary. Cells that are stably transfectedwith the recombinant enzymes express this enzyme constitutively and noinducing agent is used. This is in fundamental contrast with other cellsin which COX-2 has to be induced. Induction of COX-2 is commonlyperformed using bacterial LPS or various cytokines such asinterleukin-1β or tumor necrosis factor. Additionally, these endotoxinsand cytokines are administered at various concentrations.

[0017] Fourth, the duration of the incubation with the test agent, theCOX-2 inducing agent, or with arachidonic acid varies among differentlaboratories. These differences can influence the quantitative outcomeof the study, because the inhibition of COX-2 is time dependent.Finally, the protein concentration of the medium can vary; this is anissue for compounds that can bind avidly to plasma proteins.

[0018] An ideal assay for COX-2 selectivity would have the followingcharacteristics: (1) whole cells should be used that contain nativehuman enzymes under normal physiological control regarding expression;(2) the cells should also be target cells for the anti-inflammatory andadverse effects of the compounds; (3) COX-2 should be induced, therebysimulating an inflammatory process, rather than being constitutivelyexpressed; and (4) PG synthesis should be measured from arachidonic acidreleased from endogenous stores rather than from exogenously addedarachidonic acid.

[0019] Differences in methodology for can explain a dramatic differencein the results obtained for COX inhibition. For example, when assayedagainst the purified enzyme, ursolic acid exhibited an IC₅₀ of 130 μM,far outside of possible physiologically obtainable concentrations[Ringbom, T. et al. (1998) Ursolic acid from Plantago major, a selectiveinhibitor of cyclooxygenase-2 catalyzed prostaglandin biosynthesis. JNat Prod 61, 1212-1215]. In the RAW 264.7 murine macrophage line, Suh etal. report an IC₅₀ for ursolic acid of approximately 40 μM [Suh, N., etal. (1998) Novel triterpenoids suppress inducible nitric oxide synthase(iNOS) and inducible cyclooxygenase (COX-2) in mouse macrophages. CancerRes 58, 717-723]; and in phorbol 12-myristate 13-acetate stimulatedhuman mammary cells, the approximate median inhibitory concentration ofursolic acid was 3.0 μM [Subbaramaiah, K. et al. (2000) Ursolic acidinhibits cyclooxygenase-2 transcription in human mammary epithelialcells. Cancer Res 60, 2399-2404].

[0020] No laboratory has, as yet, developed an ideal assay for COX-2selectivity. The whole cell system most commonly used for Rx and OTCproducts is the human whole blood assay developed by the William HarveyInstitute [Warner, T. D. et al. (1999) Nonsteroid drug selectivities forcyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated withhuman gastrointestinal toxicity: a full in vitro analysis. Proc NatlAcad Sci U S A 96, 7563-7568]. To date, this assay format has developedmore data supporting clinical relevance than any other. However, newresearch in the role of constitutive expression of COX-2 in normalgastric mucosa necessitates revisiting the relevance of the use ofplatelets to model COX-1 inhibition in the absence of COX-2. Theextrapolation of gastrotoxicity from platelet studies is no longer on asound molecular basis. The validation of a human gastric mucosal cellline for establishing the potential target tissue toxicity ofcyclooxygenase inhibitors represents a critical need for the developmentof safe and effective anti-inflammatory agents.

[0021] Therefore, it would be useful to identify a composition thatwould specifically inhibit or prevent the expression of COX-2 enzymaticactivity in inflammatory cells, while having little or no effect on PGE₂synthesis in gastric mucosal cells so that these formulations could beused with no gastrointestinal upset. Furthermore, such formulationsshould allow for healing of pre-existing ulcerative conditions in thestomach.

SUMMARY OF THE INVENTION

[0022] Thus, it would be useful to identify a formulation of compoundsthat would to modulate the inflammatory response in cells. Such aformulation has widespread applications.

[0023] It would also be useful to identify a formulation of compoundsthat would inhibit expression of COX-2, inhibit prostaglandin synthesisselectively in target cells, or inhibit inflammation responseselectively in target cells. For example, it would also be useful toidentify a formulation of compounds that would specifically inhibit orprevent the synthesis of prostaglandins by COX-2 in inflammatory cellswith little or no effect on PGE₂ synthesis in gastric mucosal cells.Such a formulation, which would be useful for preserving the health ofjoint tissues, for treating arthritis or other inflammatory conditions,has not previously been discovered. Preferably, the formulations have amedian effective concentration for COX-2 inhibition in inflammatorycells that is minimally ten times greater than the median effectiveconcentration for the inhibition of PGE₂ synthesis in gastric cells. Forexample, if the median inhibitory concentration for COX-2 of a testformulation was 0.2 μg/mL in the murine macrophage RAW 264.7, theformulation would not be considered to have low potential for gastricirritancy unless the median inhibitory concentration for PGE₂ synthesisin gastric cells was equal to or greater than 2 μg/mL.

[0024] A preferred embodiment comprises compositions containing at leastone fraction isolated or derived from hops (Humulus lupulus). Examplesof fractions isolated or derived from hops are isoalpha acids, reducedisoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids,beta acids, and spent hops. Preferred compounds of fractions isolated orderived from hops, include, but are not limited to, cohumulone,adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone. Preferred compounds can also bear substituents,such as halogens, ethers, and esters.

[0025] Preferred compositions also inhibit the inducibility or activityof COX-2. Preferred compositions also can inhibit prostaglandinsynthesis selectively in target cells. Preferred compositions also caninhibit inflammation response selectively in target cells.

[0026] The compositions have widespread applications. Preferredcompositions can be useful for treating conditions, such as cancer,autoimmune diseases, inflammatory diseases, neurological diseases.Preferred compositions are also believed to be useful for treatingconditions, such as HIV-1 infections, rhinovirus infections, andcardiovascular diseases.

[0027] Preferred compositions would be useful for, but not limited to,the treatment of inflammation in a subject, and for treatment of otherinflammation-associated disorders, such as an analgesic in the treatmentof pain and headaches, or as an antipyretic for the treatment of fever.Preferred compositions would be useful to treat arthritis, including butnot limited to rheumatoid arthritis, spondyloathopathies, goutyarthritis, osteoarthritis, systemic lupus erythematosis, and juvenilearthritis.

[0028] Preferred compositions would be useful in the treatment ofasthma, bronchitis, menstrual cramps, tendonitis, bursitis, andskin-related conditions such as psoriasis, eczema, burns and dermatitis.Preferred compositions also would be useful to treat gastrointestinalconditions such as inflammatory bowel disease, Crohn's disease,gastritis, irritable bowel syndrome and ulcerative colitis and for theprevention or treatment of cancer such as colorectal cancer.

[0029] Further, preferred compositions would be useful in treatinginflammation in such diseases as vascular diseases, migraine headaches,periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease,sclerodma, rheumatic fever, type I diabetes, myasthenia gravis, multiplesclerosis, sacoidosis, nephrotic syndrome, Behchet's syndrome,polymyositis, gingivitis, hypersensitivity, swelling occurring afterinjury, myocardial ischemia and the like.

[0030] Additionally, preferred compositions would also be useful in thetreatment of ophthalmic diseases, such as retinopathies, conjunctivitis,uveitis, ocular photophobia, and of acute injury to the eye tissue.Preferred compositions would also be useful in the treatment ofpulmonary inflammation, such as that associated with viral infectionsand cystic fibrosis.

[0031] Preferred compositions would also be useful for the treatment ofcertain nervous system disorders such as cortical dementias includingAlzheimer's disease. As inhibitors of COX-2 mediated biosynthesis ofPGE₂ in inflammatory cells, these compositions would also be useful inthe treatment of allergic rhinitis, respiratory distress syndrome,endotoxin shock syndrome, atherosclerosis, and central nervous systemdamage resulting from stroke, ischemia and trauma.

[0032] Preferred embodiments further provides a composition to increasethe rate at which glucosamine or chondrotin sulfate function tonormalize joint movement or reduce the symptoms of osteoarthritis.

[0033] Preferred embodiments also provide for methods of identifyingcompositions that would specifically inhibit or prevent the synthesis ofprostaglandins by COX-2 in inflammatory cells with little or no effecton PGE₂ synthesis in gastric mucosal cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034]FIG. 1 depicts the induction of cyclooxygenase-2 and themetabolism of arachidonic acid to prostaglandins and other eicosanoidsby the cyclooxygenase enzymes. The action of non-steroidalanti-inflammatory agents is through direct inhibition of thecyclooxygenase enzymes.

[0035]FIG. 2 shows an outline of fractions and compounds that can beobtained from hops.

[0036]FIG. 3 illustrates [A] the alpha-acid genus (AA) andrepresentative species humulone (R═—CH₂CH(CH₃)₂), cohumulone(R═,—CH(CH₃)₂), and adhumulone (R═—CH(CH₃)CH₂CH₃); [B] the isoalpha acidgenus (IAA) and representative species isohumulone (R═—CH₂CH(CH₃)₂),isocohumulone (R═,—CH(CH₃)₂), and isoadhumulone (R═—CH(CH₃)CH₂CH₃); [C]the reduced isomerized isoalpha acid genus (RIAA) and representativespecies dihydro-isohumulone (R═—CH₂CH(CH₃)₂) dihydro-isocohumulone(R═,—CH(CH₃)₂), and dihydro-adhumulone (R═—CH(CH₃)CH₂CH₃); [D] thetetra-hydroisoalpha acid genus (THIAA) and representative speciestetra-hydro-isohumulone (R═—CH₂CH(CH₃)₂), tetra-hydro-isocohumulone((R═,—CH(CH₃)₂), and tetra-hydro-adhumulone (R═—CH(CH₃)CH₂CH₃); [E] andthe hexa-hydroisoalpha acid (HHIAA) genus with representative specieshexa-hydro-isohumulone (R═—CH₂CH(CH₃)₂) hexa-hydro-isocohumulone(R═,—CH(CH₃)₂), and hexa-hydro-adhumulone (R═—CH(CH₃)CH₂CH₃).

[0037]FIG. 4 are representative immunoblots demonstrating constitutiveCOX-1 and COX-2 expression in AGS human gastric mucosal cells. The AGShuman gastric cell line was cultured in 6-well plates at 37° C. with 5%CO₂ in a humidified incubator for 24 hours. Cells were lysed on ice inlysis buffer and protein concentration determined. Fifty μg of celllysate were solubilized, fractionated on a 10% polyacrylamide gelcontaining sodium dodecylsulfate (SDS), and transferred onto anitrocellulose membrane. The membranes were incubated in a blockingbuffer and then incubated with the respective primary antibody for 1 hat room temperature. Following primary antibody incubation, the blotswere washed three times with Tris-buffered saline and then incubatedwith the secondary antibody for 1 h. Protein bands were visualized usingenhanced chemiluminescence.

[0038]FIG. 5 illustrates the induction of PGE₂ synthesis by miteallergen in A549 pulmonary cells treated for 24 hours.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0039] The present invention relates to the discovery that that asupragenus of components isolated or derived from hops and othercompounds result in tissue-specific or cell-specific inhibition of COX-2expression. Importantly, these compounds are not believed to directlyinhibit COX-2 or other enzymes with the prostaglandin synthesis pathway.Preferred embodiments provide compositions and methods for inhibitingCOX-2 expression, inhibiting prostanglandin synthesis selectively intarget tissues or cells, or inhibiting inflammation response selectivelyin target tissues or cells.

[0040] A preferred embodiment comprises compositions containingfractions or compounds isolated or derived from hops. Examples offractions isolated or derived from hops are alpha acids, isoalpha acids,reduced isoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalphaacids, beta acids, and spent hops. Preferred compounds of the fractionsisolated or derived from hops can be represented by a supragenus below:

[0041] (Supragenus),

[0042] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; wherein R″ is selected fromthe group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; andwherein R, T, X, and Z are independently selected from the groupconsisting of H, F, Cl, Br, I, and a orbital, with the proviso that ifone of R, T, X, or Z is a π orbital, then the adjacent R, T, X, or Z isalso a π orbital, thereby forming a double bond.

[0043] Other preferred compounds of the fractions isolated or derivedfrom hops can be represented by a genus below:

[0044] (Genus A),

[0045] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; and wherein R″ is selectedfrom the group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

[0046] Other preferred compounds of the fractions isolated or derivedfrom hops can be represented by a genus below:

[0047] (Genus B),

[0048] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; and wherein R″ is selectedfrom the group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

[0049] Examples of preferred compounds of an ingredient isolated orderived from hops, include, but are not limited to, humulone,cohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone. The preferred compounds can bear substituents, asshown in the formula above.

[0050] As used herein, the term “dietary supplement” refers tocompositions consumed to affect structural or functional changes inphysiology. The term “therapeutic composition” refers to any compoundsadministered to treat or prevent a disease.

[0051] As used herein, the term “effective amount” means an amountnecessary to achieve a selected result. Such an amount can be readilydetermined without undue experimentation by a person of ordinary skillin the art.

[0052] As used herein, the term “substantial” means being largely butnot wholly that which is specified.

[0053] As used herein, the term “COX inhibitor” refers to a compositionof compounds that is capable of inhibiting the activity or expression ofCOX-2 enzymes or is capable of inhibiting or reducing the severity,including pain and swelling, of a severe inflammatory response.

[0054] As used herein, the terms “derivatives” or a matter “derived”refer to a chemical substance related structurally to another substanceand theoretically obtainable from it, i.e. a substance that can be madefrom another substance. Derivatives can include compounds obtained via achemical reaction.

[0055] As used herein, the term “inflammatory cell” refers to thosecellular members of the immune system, for example B and T lymphocytes,neutrophils or macrophages involved in synthesis of prostaglandins inresponse to inflammatory signals such as interleukins, tumor necrosisfactor, bradykinin, histamine or bacterial-derived components.

[0056] As used herein, the term “target cells” refers to that cellpopulation in which the inhibition of PGE₂ or other prostaglandinsynthesis is desired, such as inflammatory cells, tumor cells, orpulmonary cells. Alternatively, “non-target cells” refers to that cellpopulation in which the inhibition of PGE₂ or other prostaglandinsynthesis is not desired, such as the gastric mucosal, neural or renalcells.

[0057] As used herein, the term “hop extract” refers to the solidmaterial resulting from (1) exposing a hops plant product to a solvent,(2) separating the solvent from the hops plant products, and (3)eliminating the solvent.

[0058] As used herein, the term “solvent” refers to a liquid of aqueousor organic nature possessing the necessary characteristics to extractsolid material from the hop plant product. Examples of solvents wouldinclude, but not limited to, water, steam, superheated water, methanol,ethanol, hexane, chloroform, liquid CO₂, liquid N₂ or any combinationsof such materials.

[0059] As used herein, the term “CO₂ extract” refers to the solidmaterial resulting from exposing a hops plant product to a liquid orsupercritical CO₂ preparation followed by the removing the CO₂.

[0060] As used herein, the term “spent hops” refers to the solid andhydrophilic residue from extract of hops.

[0061] As used herein, the term “alpha acid” refers to compounds refersto compounds collectively known as humulones and can be isolated fromhops plant products including, among others, humulone, cohumulone,adhumulone, hulupone, and isoprehumulone.

[0062] As used herein, the term “isoalpha acid” refers to compoundsisolated from hops plant products and subsequently have been isomerized.The isomerization of alpha acids can occur thermally, such as boiling.Examples of isoalpha acids include, but are not limited to, isohumulone,isocohumulone, and isoadhumulone.

[0063] As used herein, the term “reduced isoalpha acid” refers to alphaacids isolated from hops plant product and subsequently have beenisomerized and reduced, including cis and trans forms. Examples ofreduced isoalpha acids (RIAA) include, but are. not limited to,dihydro-isohumulone, dihydro-isocohumulone, and dihydro-adhumulone.

[0064] As used herein, the term “tetra-hydroisoalpha acid” refers to acertain class of reduced isoalpha acid. Examples of tetra-hydroisoalphaacid (THIAA) include, but are not limited to, tetra-hydro-isohumulone,tetra-hydro-isocohumulone and tetra-hydro-adhumulone:

[0065] As used herein, the term “hexa-hydroisoalpha acid” refers to acertain class of reduced isoalpha acid. Examples of hexa-hydroisoalphaacids (HHIAA) include, but are not limited to, hexa-hydro-isohumulone,hexa-hydro-isocohumulone and hexa-hydro-adhumulone.

[0066] As used herein, the term “beta-acid fraction” refers to compoundscollectively known as lupulones including, among others, lupulone,colupulone, adlupulone, tetrahydroisohumulone, and hexahydrocolupulone.

[0067] As used herein, the term “essential oil fraction” refers to acomplex mixture of components including, among others, myrcene,humulene, beta-caryophyleen, undecane-2-on, and 2-methyl-but-3-en-ol.

[0068] As used herein, “conjugates” of compounds means compoundscovalently bound or conjugated to a member selected from the groupconsisting of mono- or di-saccharides, amino acids, sulfates, succinate,acetate, and glutathione. Preferably, the mono- or di- saccharide is amember selected from the group consisting of glucose, mannose, ribose,galactose, rhamnose, arabinose, maltose, and fructose.

[0069] Hops

[0070] Hop extraction in one form or another goes, back over 150 yearsto the early nineteenth century when extraction in water and ethanol wasfirst attempted. Even today an ethanol extract is available in Europe,but by far the predominant extracts are organic solvent extracts(hexane) and CO₂ extracts (supercritical and liquid). CO₂ (typically at60 bars pressure and 50 to 10° C.) is in a liquid state and is arelatively mild, non-polar solvent highly specific for hop soft resinsand oils. Beyond the critical point, typically at 300 bars pressure and60° C., CO₂ has the properties of both a gas and a liquid and is a muchstronger solvent. The composition of the various extracts is compared inTable 2. TABLE 2 Hop Extracts (Percent W/W) Super- Component HopsOrganic Solvent Critical CO₂ Liquid CO₂ Total resins 12-20 15-60  75-9070-95 Alpha-acids  2-12 8-45 27-55 30-60 Beta-acids  2-10 8-20 23-3315-45 Essential oils 0.5-1.5 0-5  1-5  2-10 Hard resins 2-4 2-10  5-11None Tannins  4-10 0.5-5   0.1-5   None Waxes 1-5 1-20  4-13  0-10 Water 8-12 1-15 1-7 1-5

[0071] At its simplest, hop extraction involves milling, pelleting andre-milling the hops to spread the lupulin, passing a solvent through apacked column to collect the resin components and finally, removal ofthe solvent to yield a whole or “pure” resin extract.

[0072] The main organic extractants are strong solvents and in additionto virtually all the lupulin components, they extract plant pigments,cuticular waxes, water and water-soluble materials.

[0073] Supercritical CO₂ is more selective than the organic solvents andextracts less of the tannins and waxes and less water and hencewater-soluble components. It does extract some of the plant pigmentslike chlorophyll but rather less than the organic solvents do. LiquidCO₂ is the most selective solvent used commercially for hops and henceproduces the most pure whole resin and oil extract. It extracts hardlythe hard resins or tannins, much lower levels of plant waxes, no plantpigments and less water and water-soluble materials.

[0074] As a consequence of this selectivity and the milder solventproperties, the absolute yield of liquid CO₂, extract per unit weight ofhops is less than when using the other mentioned solvents. Additionally,the yield of alpha acids with liquid CO₂ (89-93%) is lower than that ofsupercritical CO₂ (91-94%) or the organic solvents (93-96%). Followingextraction there is the process of solvent removal, which for organicsolvents involves heating to cause volatilization. Despite this, traceamounts of solvent do remain in the extract. The removal of CO₂,however, simply involves a release of pressure to volatize the CO₂.

[0075] As shown in FIG. 2, hops CO₂ extracts can be fractionated intocomponents, including hops oils, beta acids, and alpha acids. Hops oilsinclude, but not limited to, humulene, beta-caryophyllene, mycrene,famescene, gamma-cadinene, alpha-selinene, and alpha-cadinene. Betaacids include, but are not limited to, lupulone, colupulone, adlupulone,tetrahydroisohumulone, and hexahydrocolupulone, collectively known aslupulones. Beta acids can be isomerized and reduced. Beta acids arereduced to give tetra-beta acids. Alpha acids include, but are notlimited to, humulone, cohumulone, adhumulone, hulupone, andisoprehumulone. Alpha acids can be isomerized to give isoalpha acids.Iso-alpha acids can be reduced to give reduced-isoalpha acids,tetra-hydroisoalpha acids, and hexa-hydroisoalpha acids.

[0076] A preferred embodiment comprises compositions containingfractions or compounds isolated or derived from hops. Examples offractions isolated or derived from hops are alpha acids, isoalpha acids,reduced isoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalphaacids, beta acids, and spent hops. Preferred compounds of the fractionsisolated or derived from hops can be represented by a supragenus below:

[0077] (Supragenus),

[0078] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; wherein R″ is selected fromthe group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; andwherein R, T, X, and Z are independently selected from the groupconsisting of H, F, Cl, Br, I, and π orbital, with the proviso that ifone of R, T, X, or Z is a π orbital, then the adjacent R, T, X, or Z isalso a π orbital, thereby forming a double bond.

[0079] Other preferred compounds of the fractions isolated or derivedfrom hops can be represented by a genus below:

[0080] (Genus A),

[0081] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; and wherein R″ is selectedfrom the group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

[0082] Other preferred compounds of the fractions isolated or derivedfrom hops can be represented by a genus below:

[0083] (Genus B),

[0084] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; and wherein R″ is selectedfrom the group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

[0085] As shown in FIG. 3, examples of preferred compounds of aningredient isolated or derived from hops, include, but are not limitedto, humulone, cohumulone, adhumulone, isohumulone, isocohumulone,isoadhumulone, dihydro-isohumulone, dihydro-isocohumulone,dihydro-adhumulone, tetrahydro-isohumulone, tetrahydro-isocohumulone,tetrahydro-adhumulone, hexahydro-isohumulone, hexahydro-isocohumulone,and hexahydro-adhumulone. The preferred compounds can bear substituents,as shown in the formula above.

[0086] The identification of humulone from hops extract as an inhibitorof bone resorption is reported in Tobe, H. et al. 1997. (Bone resorptionInhibitors from hop extract. Biosci. Biotech. Biochem 61(1)158-159.)Later studies by the same group characterized the mechanism of action ofhumulone as inhibition of COX-2 gene transcription following TNFalphastimulation of MC3T3, El cells [Yamamoto, K. 2000. Suppression ofcyclooxygenase-2 gene transcription by humulon of beer hop extractstudied with reference to the glucocorticoid receptor. FEBS Letters465:103-106]. The authors concluded that the action of humulone (alsohumulon) was similar to that of glucocorticoids, but that humulone didnot function through the glucocorticoid receptor. While these resultsestablish that humulone inhibits PGE₂ synthesis in MC3T3 cells(osteoblasts) at the gene level, one skilled in the art would not assumethat these results would necessarily occur in immune inflammatory cellsor other cell lines. Example 5 herein demonstrates the high degree oftissue selectivity of hops compounds and derivatives.

[0087] The preferred embodiments also provide compositions and methodsfor inhibiting expression of COX-2, inhibiting synthesis ofprostaglandins selectively in target cells, and inhibiting inflammatoryresponse selectively in target cells. Preferred methods comprise a stepof administering to a mammal a composition of the preferred embodiments.Preferred embodiments comprise a fraction isolated or derived from hops.A certain composition comprises alpha acids, isoalpha acids, reducedisoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids,beta acids, or spent hops from hops extract or derivatives thereof.Preferred compounds of the fractions isolated or derived from hops canbe represented by a supragenus below:

[0088] (Supragenus),

[0089] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; wherein R″ is selected fromthe group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; andwherein R, T, X, and Z are independently selected from the groupconsisting of H, F, Cl, Br, I and π orbital, with the proviso that ifone of R, T, X, or Z is a π orbital, then the adjacent R, T, X, or Z isalso a π orbital, thereby forming a double bond. Other preferredcompounds of the fractions isolated or derived from hops can berepresented by a genus below:

[0090] (Genus A),

[0091] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; and wherein R″ is selectedfrom the group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.Other preferred compounds of the fractions isolated or derived from hopscan be represented by a genus below:

[0092] (Genus B),

[0093] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; and wherein R″ is selectedfrom the group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.The preferred embodiments contemplate compositions comprising beta acidsor isomerized or reduced beta acids. Preferably, the alpha acid,isoalpha acid, reduced isoalpha acid, tetra-hydroisoalpha acid,hexa-hydroisoalpha acid, beta acid, or spent hops of the preferredembodiments is made from hops extract. More preferably, the alpha acid,isoalpha acid, reduced isoalpha acid, tetra-hydroisoalpha acid,hexa-hydroisoalpha acid, beta acid, or spent hops of the preferredembodiments is made from CO₂ extract of hops.

[0094] Compositions

[0095] The preferred compositions can function to specifically inhibitCOX-2 expression, to inhibit prostaglandin synthesis selectively intarget cells, or to inhibit inflammation response selectively in targetcells. Preferred embodiments include compositions containing fractionsor compounds isolated or derived from hops.

[0096] A preferred embodiment comprises compositions containingfractions or compounds isolated or derived from hops. Examples offractions isolated or derived from hops are alpha acids, isoalpha acids,reduced isoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalphaacids, beta acids, and spent hops. Preferred compounds of the fractionsisolated or derived from hops can be represented by a supragenus below:

[0097] (Supragenus),

[0098] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; wherein R″ is selected fromthe group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; andwherein R, T, X, and Z are independently selected from the groupconsisting of H, F, Cl, Br, I and π orbital, with the proviso that ifone of R, T, X, or Z is a π orbital, then the adjacent R, T, X, or Z isalso a π orbital, thereby forming a double bond.

[0099] Other preferred compounds of the fractions isolated or derivedfrom hops can be represented by a genus below:

[0100] (Genus A),

[0101] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; and wherein R″ is selectedfrom the group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

[0102] Other preferred compounds of the fractions isolated or derivedfrom hops can be represented by a genus below:

[0103] (Genus B),

[0104] wherein R′ is selected from the group consisting of carbonyl,hydroxyl, OR, and OCOR, wherein R is alkyl; and wherein R″ is selectedfrom the group consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.

[0105] Examples of preferred compounds of an ingredient isolated orderived from hops, include, but are not limited to, humulone,cohumulone, adhumulone, isohumulone, isocohumulone, isoadhumulone,dihydro-isohumulone, dihydro-isocohumulone, dihydro-adhumulone,tetrahydro-isohumulone, tetrahydro-isocohumulone, tetrahydro-adhumulone,hexahydro-isohumulone, hexahydro-isocohumulone, andhexahydro-adhumulone. The preferred compounds can bear substituents, asshown in the formula above.

[0106] Other embodiments relate to combinations of components. Thepreferred compositions can also function synergistically to specificallyinhibit COX-2 expression, to inhibit prostaglandin synthesis selectivelyin target cells, or to inhibit inflammation response selectively intarget cells.

[0107] Dosage

[0108] The selected dosage level will depend upon activity of theparticular composition, the route of administration, the severity of thecondition being treated or prevented, and the condition and priormedical history of the patient being treated. However, it is within theskill of the art to start doses of the composition at levels lower thanrequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. If desired,the effective daily dose may be divided into multiple doses for purposesof administration, e.g., two to four separate doses per day. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors including body weight,general health, diet, time and route of administration, combination withother compositions and the severity of the particular condition beingtreated or prevented.

[0109] Preferred embodiments include delivering an effective amount ofhops fractions, hops compounds, or hops derivatives alone or with incombination with other active ingredients. Preferably, a daily dose ofpreferred compositions would be formulated to deliver about 0.5 to10,000 mg of alpha acid, isoalpha acid, reduced isoalpha acid,tetra-hydroisoalpha acid, hexa-hydroisoalpha acid, beta acid, or spenthops per day. More preferably, an effective daily dose of preferredcompositions would be formulated to deliver about 50 to 7500 mg of alphaacids, isoalpha acid, reduced isoalpha acid, tetra-hydroisoalpha acid,hexa-hydroisoalpha acid, beta acid, or spent hops per day. Preferably,the effective daily dose is administered once or twice a day. A certainembodiment provides a composition comprising about 0.5 to 500 mg ofisoalpha acid or reduced isoalpha acid, more preferably about 50 to 300mg of isoalpha acid or reduced isoalpha acid per day. Another certainembodiment provides a composition comprising about 10 to 3000 mg ofreduced isoalpha acid, tetra-hydroisoalpha acid, or hexa-hydroisoalphaacid per day, more preferably about 50 to 2000 mg of reduced isoalphaacid, tetra-hydroisoalpha acid, or hexa-hydroisoalpha acid per day. Yetanother certain embodiment provides a composition comprising about 50 to7500 mg of spent hops per day, preferably about 100 to 6000 mg of spenthops per day.

[0110] A composition of preferred embodiments for topical applicationwould contain about 0.001 to 10 weight percent, preferably about 0.1 to1 weight percent of a hops derivative. Preferred embodiments wouldproduce serum concentrations in the ranges of about 0.0001 to 10 μM,preferably about 0.01 to 1 μM of a fraction isolated or derived fromhops or conjugate thereof.

[0111] Applications of Preferred Compositions

[0112] As stated previously, the generally held concept (COX dogma) isthat COX-1 is expressed constitutively in most tissues whereas COX-2 isthe inducible enzyme triggered by pro-inflammatory stimuli includingmitogens, cytokines and bacterial lipopolysaccharide (LPS) in cells invitro and in inflamed sites in vivo. Based primarily on such differencesin expression, COX-1 has been characterized as a housekeeping enzyme andis thought to be involved in maintaining physiological functions such ascytoprotection of the gastric mucosa, regulation of renal blood flow,and control of platelet aggregation. COX-2 is considered to mainlymediate inflammation, although constitutive expression is found inbrain, kidney and the gastrointestinal tract. Therefore, it would bedesirable to down-regulate expression of COX-2 tissue-specifically orcell-specifically. Examples of target cells include, but are not limitedto, inflammatory cells, pulmonary cells, and tumor cells. Examples ofnontarget cells include, but are not limited to, gastric mucosal,neural, and renal cells.

[0113] The compositions have widespread applications. Preferredcompositions can be useful for treating conditions, such as cancer,autoimmune diseases, inflammatory diseases, neurological diseases.Preferred compositions are also believed to be useful for treatingconditions, such as HIV-1 infections, rhinovirus infections, andcardiovascular diseases.

[0114] Preferred embodiments would be useful for, but not limited to anumber of inflammatory conditions. Thus, preferred embodiments includetreatment of inflammation in a subject, and treatment of otherinflammation-associated disorders, such as, as an analgesic in thetreatment of pain and headaches, or as an antipyretic for the treatmentof fever. Additional examples of such preferred embodiments would beuseful to treat arthritis, including but not limited to rheumatoidarthritis, spondyloathopathies, gouty arthritis, osteoarthritis,systemic lupus erythematosis, and juvenile arthritis. Such preferredembodiments would be useful in the treatment of asthma, bronchitis,menstrual cramps, tendonitis, bursitis, and skin related conditions suchas psoriasis, eczema, burns and dermatitis. Preferred embodiments alsowould be useful to treat gastrointestinal conditions such asinflammatory bowel disease, Crohn's disease, gastritis, irritable bowelsyndrome and ulcerative colitis and for the prevention or treatment ofcancer such as colorectal cancer. Preferred embodiments would be usefulin treating inflammation in such diseases as vascular diseases, migraineheadaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin'sdisease, sclerodma, rheumatic fever, type I diabetes, myasthenia gravis,multiple sclerosis, sacoidosis, nephrotic syndrome, Behchet's syndrome,polymyositis, gingivitis, hypersensitivity, swelling occurring afterinjury, myocardial ischemia and the like.

[0115] Preferred embodiments would also be, useful in the treatment ofophthalmic diseases, such as retinopathies, conjunctivitis, uveitis,ocular photophobia, and of acute injury to the eye tissue. Preferredembodiments would also be useful in the treatment of pulmonaryinflammation, such as that associated with viral infections and cysticfibrosis. Preferred embodiments would also be useful in the treatment ofasthma. Preferred embodiments would also be useful for the treatment ofcertain nervous system disorders such as cortical dementias includingAlzheimer's disease. Preferred embodiments are useful asanti-inflammatory agents, such as for the treatment of arthritis, withthe additional benefit of having significantly less harmful sideeffects. As inhibitors of COX-2 mediated biosynthesis of PGE₂, thesecompositions would also be useful in the treatment of allergic rhinitis,respiratory distress syndrome, endotoxin shock syndrome,atherosclerosis, and central nervous system damage resulting fromstroke, ischemia and trauma. The preferred embodiments would also beuseful for the treatment of fibromyalgia.

[0116] Since COX-2 can also play a role in the regulation ofosteoblastic function, preferred embodiments can also be useful fortreating and preventing osteoporosis. Kanematsu et al. (J Bone Miner Res1997 Nov;12(11):1789-96.) discloses that interleukin 1 (IL-1) and tumornecrosis factor alpha (TNF-alpha) have been implicated in thepathogenesis of osteoporosis. These proinflammatory cytokines induceboth COX-2 and nitric oxide synthase (iNOS) with the release of PGE₂ andNO, respectively. They determined the interaction between COX and NOSpathways and their role in the regulation of osteoblastic function inMC3T3-E1 cells.

[0117] According to preferred embodiments, the animal may be a memberselected from the group consisting of humans, non-human primates, dogs,cats, birds, horses, ruminants or other warm blooded animals. Preferredembodiments are directed primarily to the treatment of human beings.Administration can be by any method available to the skilled artisan,for example, by oral, topical, transdermal, transmucosal, or parenteralroutes.

[0118] Besides being useful for human treatment, preferred embodimentsare also useful for treatment of other animals, including horses, dogs,cats, birds, sheep, pigs, etc. A certain formulation for the treatmentof inflammation would inhibit the induction and activity of COX-2 withlittle effect on the synthesis of PGE₂ in the gastric mucosa.Historically, the NSAIDs used for treatment of inflammation lacked thespecificity of inhibiting COX-2 without affecting PGE₂ synthesis ingastric mucosal cells. Therefore, these drugs irritated and damaged thegastrointestinal system when used for extended periods.

[0119] Formulations

[0120] Preferred compositions can be administered in the form of adietary supplement or therapeutic composition. The compositions may beadministered orally, topically, transdermally, transmucosally,parenterally, etc., in appropriate dosage units, as desired.

[0121] Preferred compositions for dietary application may includevarious additives such as other natural components of intermediarymetabolism, vitamins and minerals, as well as inert ingredients such astalc and magnesium stearate that are standard excipients in themanufacture of tablets and capsules. For example, one embodimentcomprises active ingredients of preferred compositions in combinationwith glucosamine or chondrotin sulfate.

[0122] As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, isotonic andabsorption delaying agents, sweeteners and the like. Thesepharmaceutically acceptable carriers may be prepared from a wide rangeof materials including, but not limited to, diluents, binders andadhesives, lubricants, disintegrants, coloring agents, bulking agents,flavoring agents, sweetening agents and miscellaneous materials such asbuffers and absorbents that may be needed in order to prepare aparticular therapeutic composition. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in preferred compositions is contemplated.In one embodiment, talc, and magnesium stearate are included in theformulation. Other ingredients known to affect the manufacture of thiscomposition as a dietary bar or functional food can include flavorings,sugars, amino-sugars, proteins and/or modified starches, as well as fatsand oils.

[0123] Dietary supplements, lotions or therapeutic compositions ofpreferred embodiments can be formulated in any manner known by one ofskill in the art. In one embodiment, the composition is formulated intoa capsule or tablet using techniques available to one of skill in theart. In capsule or tablet form, the recommended daily dose for an adulthuman or animal would preferably be contained in one to six capsules ortablets. However, preferred compositions can also be formulated in otherconvenient forms, such as an injectable solution or suspension, a spraysolution or suspension, a lotion, gum, lozenge, food or snack item.Food, snack, gum or lozenge items can include any ingestible ingredient,including sweeteners, flavorings, oils, starches, proteins, fruits orfruit extracts, vegetables or vegetable extracts, grains, animal fats orproteins. Thus, preferred compositions can be formulated into cereals,snack items such as chips, bars, gumdrops, chewable candies or slowlydissolving lozenges. Preferred embodiments contemplate treatment of alltypes of inflammation-based diseases, both acute and chronic. Preferredformulations reduce the inflammatory response and thereby promoteshealing of, or prevents further damage to, the affected tissue. Apharmaceutically acceptable carrier can also be used in the preferredcompositions and formulations.

[0124] Assay using AGS Cell Line

[0125] The Kuhrts patent application referenced previously attempts toidentify therapeutic components based on the Modified Whole Blood/CellAssay of T. D. Warner et al., Nonsteroid drug selectivities forcyclooxygenase-1 rather than cyclooxygenase-2 are associated with humangastrointestinal toxicity: A full in vitro analysis, Proc. Natl. Sci.USA 96:7563-68(1999) in paragraph [0046]. When tested according to thisprocedure, hops extracts do not yield IC₅₀ values in the necessary μg/mLrange, since they are not direct inhibitors of COX-2. This lack ofdirect inhibition of COX-2 was demonstrated by Tobe, H. et al. 1997.(Bone resorption Inhibitors from hop extract. Biosci. Biotech. Biochem61(1)158-159) using purified COX-2 enzyme. Similarly, Example 4 of thisapplication demonstrates that, when tested according to the ModifiedWhole Blood/Cell Assay, hops compounds and derivatives produce medianinhibitory concentrations greater than 25 μg/mL. Such high medianinhibitory concentrations are pharmacologically unsuitable. Therefore,the Modified Whole Blood Assay as described by Warner is an invalidprocedure for formulating potentially therapeutically effectivecombinations containing hops or hops derivatives.

[0126] The discovery of COX-2 has made possible the design of drugs thatreduce inflammation without removing the protective PGs in the stomachand kidney made by COX-1. One of our approaches is to screencompositions of the preferred embodiments using in vitro animal cells toassess COX-2 and COX-1 inhibitory activity employing PGE₂, which hascytoprotective actions and play a role in maintaining the integrity ofthe gastrointestinal mucosa, as an endpoint. Secondarily, different celltypes are used to confirm results. The screening process would indicatecompositions that have specific COX-2 activity and limited COX-1inhibition. Compositions of preferred embodiments can be tested in twocell types: 1) human pulmonary cells or other cell line to determine andidentify optimal amounts and ratios for compositions comprising morethan one component; and 2) human gastric epithelial cells (AGS cellline), a gastrointestinal tract cell line and a model system forassessing toxicity which is typically related to inhibition of COX-1which is required for wound healing (such as ulcers). Hence,compositions of preferred embodiments that can inhibit COX-2 or COX-2induction can be screened by selecting compositions that have low or noactivity in AGS cells and good activity in human pulmonary cells orother cell line.

[0127] The description below is of specific examples setting forthpreferred embodiments and are not intended to limit the scope.

EXAMPLE 1 AGS Gastric Mucosal Cells Constitutively Express BothCyclooxygenase-1 and Cyclooxygenase-2

[0128] Summary—This example demonstrates that the AGS human gastricmucosal cell line, possessing constitutive expression of COX-1 andCOX-2, has excellent potential to serve as a model for assessing thegastrointestinal toxicity of cyclooxygenase-inhibiting compounds.

[0129] Equipment used in this example included: an OHAS Model #E01140analytical balance, a Forma Model #F1214 biosafety cabinet (Marietta,Ohio), various pipettes to deliver 0.1 to 100 μL (VWR, Rochester, N.Y.),a cell hand tally counter (VWR Catalog #23609-102, Rochester, N.Y.), aForma Model #F3210 CO₂ incubator (Marietta, Ohio), a hemacytometer(Hausser Model #1492, Horsham, Pa.), a Leica Model #DM IL invertedmicroscope (Wetzlar, Germany), a PURELAB Plus Water Polishing System(U.S. Filter, Lowell, Mass.), a 4° C. refrigerator (Forma Model #F3775,Marietta, Ohio), a vortex mixer (VWR Catalog #33994-306, Rochester,N.Y.), and a 37° C. water bath (Shel Lab Model #1203, Cornelius, Oreg.).

[0130] Chemicals and reagents—Prostaglandin E₂ EIA kit Monoclonal waspurchased from Cayman Chemical (Ann Arbor, Mich.). Anti-COX-1 andanti-COX-2 rabbit polyclonal antisera were obtained from UpstateBiotechnology (CITY, N.Y.); donkey anti-goat IgG-HRP was procured fromSanta Cruz Biotechnology (City, Calif.). Heat inactivated Fetal BovineSerum (FBS-HI Cat. #35-011CV), and Dulbeco's Modification of Eagle'sMedium (DMEM Cat #10-013CV) was purchased from Mediatech (Herndon, Va.).All standard reagents were obtained from Sigma (St. Louis, Mo.) and werethe purest commercially available.

[0131] Cell Culture—The human gastric mucosal cell line AGS was obtainedfrom the American Type Culture Collection (Manassas, Va.) andsub-cultured according to the instructions of the supplier. The cellswere routinely cultured at 37° C. with 5% CO₂ in RPMI 1640 containing10% FBS, with 50 units penicillin/mL, 50 μg streptomycin/mL, 5% sodiumpyruvate, and 5% L-glutamine. Exponentially growing cells were seededinto 6-well plates and grown to confluence. A 20 μL aliquot of thesupernatant media was sampled for determination of PGE₂ content. Cellswere then washed in PBS, scraped and lysed for immunoblotting.

[0132] Protein assay—Protein concentrations of cell lysates weredetermined using the NanoOrange Protein Quantitation Kit with bovineserum albumin as the standard (Molecular Probes, Eugene, Oreg.)according to the procedure supplied by the manufacturer. Fluorescencewas determined using a Packard FluoroCount, Model BF 10000 fluorometerwith the excitation filter set at 485 nm and emission filter set at 570nm using Packard PlateReader version 3.0 software. The I-Smart programprovided with the Packard PlateReader was used to calculate the proteinconcentration.

[0133] Immunoblotting—Western blotting of COX-1 and COX-2 was performedusing PAGErTM Gold Precast Gels (Bio Whittaker Molecular Applications(Rockland, Me.). AGS cell lysates containing approximately 60 μg proteinwere loaded with Laemmli Sample Buffer into the wells of the gel in atotal volume of 30 μL. The vertical minigel electrophoresis chamberswere made by Savant Instruments Inc. (Holbrook, N.Y.), model MV 120.Gels were run at 40 mA/plate (constant current) at room temperatureuntil the bromophenol blue stain reached the bottom of the gel, aboutone h. Gels were then blotted on the polyvinyl fluoride transfermembranes (Pall Corporation, Ann Arbor, Mich.), overnight, at 500 mA and4° C. Precision Protein Standard molecular weight markers, unstained,broad range (BioRad, Hercules, Calif.) were used. The BioWest™ Extendedduration chemiluminescent substrate, a non-isotopic, horseradishperoxidase substrate kit for Western blot detection (Biolmaging Systems,Upland, Calif.) was used for protein visualization. Images of westernblots were acquired using a UVP Epi Chemi II Darkroom (BiolmagingSystems), analyzed and enhanced by LabWorks™ Image Acquisition andAnalysis Software (BioImaging Systems).

[0134] PGE₂ assay—A commercial, non-radioactive procedure forquantification of PGE₂ was employed (Caymen Chemical, Ann Arbor, Mich.)and the recommended procedure of the manufacturer was used withoutmodification. Briefly, 25 μL of the medium, along with a serial dilutionof PGE₂ standard samples, were mixed with appropriate amounts ofacetylcholinesterase-labeled tracer and PGE₂ antiserum, and incubated atroom temperature for 18 h. After the wells were emptied and rinsed withwash buffer, 200 μL of Ellman's reagent containing substrate foracetylcholinesterase were added. The reaction was carried out on a slowshaker at room temperature for 1 h and the absorbance at 415 nm wasdetermined. The PGE₂ concentration was represented as picograms per) 10⁵cells.

[0135] Results—As seen in FIG. 4, the AGS cell line constitutivelyexpresses both COX-1 and COX-2, with COX-1 expression approximately4-times greater than COX-2 expression. PGE₂ synthesis in AGS cells over18 h was 660 pg/10⁵ cells. Thus, this example demonstrates that the AGShuman gastric mucosal cell line, possessing constitutive expression ofCOX-1 and COX-2, has excellent potential to serve as a model forassessing the gastrointestinal toxicity of cyclooxygenase-inhibitingcompounds.

[0136] In the past, the classical COX-2 hypothesis has downplayed therole of COX-2 expression in the gastrointestinal mucosa. While in normalgastric mucosa COX-1 is the predominant COX isozyme, as demonstrated inthis example and in the literature, there is increasing evidence thatdetectable amount of COX-2 mRNA and protein are both constitutivelyexpressed and inducible in specific locations of the gastric mucosa inboth animals and humans [Halter, F., et al. (2001) Cyclooxygenase2-implications on maintenance of gastric mucosal integrity and ulcerhealing: controversial issues and perspectives. Gut 49, 443-453]. Recentstudies in rats have shown that whereas selective inhibition of COX-1 orCOX-2 is not ulcerogenic, combined inhibition of both COX-1 and COX-2induces severe lesions in the stomach and small intestine comparablewith the effects of NSAID such as indomethacin. This observationsuggests an important contribution of COX-2 to the maintenance ofgastrointestinal mucosal integrity.

EXAMPLE 2 Inhibition of PGE₂ Synthesis in Gastric Mucosal Cells byNonsteroidal Anti-Inflammatory Drugs

[0137] Summary—This example illustrates that inhibition of PGE₂synthesis in AGS gastric cells by NSAIDs correlates with their observedclinical gastric irritation.

[0138] Chemicals—Rofecoxib and celexocib were obtained.Diisofluorophosphate (DIFP), nimensulide, ibuprofen, salicylic acid,aspirin, indomethacin and acetaminophen were purchased from Sigma (St.Louis, Mo.). All other chemicals were obtained from suppliers asdescribed in Example 1.

[0139] Cells—A549 (human pulmonary epithelial) and AGS cells (humangastric mucosa) were obtained from the American Type Culture Collection(Manassas, Va.) and sub-cultured according to the instructions of thesupplier. The cells were routinely cultured at 37° C. with 5% CO₂ inRPMI 1640 containing-10% FBS, with 50 units penicillin/mL, 50 μgstreptomycin/mL, 5% sodium pyruvate, and 5% L-glutamine. On the day ofthe experiments, exponentially growing cells were harvested and washedwith serum-free RPMI 1640.

[0140] The log phase A549 and AGS cells were plated at 8×10⁴ cells perwell in 0.2 mL growth medium per well in a 96-well tissue culture plate.For the determination of PGE₂ inhibition by the test compounds in A549cells, the procedure of Warner et al., also known as the WHMA-COX-2protocol [Warner, T. D., et al. (1999) Nonsteroid drug selectivities forcyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated withhuman gastrointestinal toxicity: a full in vitro analysis. Proc NatlAcad Sci U S A 96, 7563-7568.] was followed with no modifications.Briefly, 24 hours after plating of the A549 cells, interleukin-1β (10ng/mL) was added to induce the expression of COX-2. After 24 hr, thecells were washed with serum-free RPMI 1640 and the test materials,dissolved in DMSO and serum-free RPMI, were added to the wells toachieve final concentrations of 25, 5.0, 0.5 and 0.05 μg/mL. Eachconcentration was run in duplicate. DMSO was added to the control wellsin an equal volume to that contained in the test wells. Sixty minuteslater, A23187 (50 μM) was added to the wells to release arachidonicacid. Twenty-five μL of media were sampled from the wells 30 minuteslater for PGE₂ determination.

[0141] Non-stimulated AGS cells were used in these studies. Twenty-fourhours after plating in the 96-well microtiter plates, the cells werewashed with serum-free RPMI 1640 and the test materials, dissolved inDMSO and serum-free RPMI, were added to the wells to achieve finalconcentrations of 25, 5.0, 0.5 and 0.05 μg/mL. Each concentration wasrun in duplicate. DMSO was added to the control wells in an equal volumeto that contained in the test wells. Sixty minutes later, arachidonicacid was added to the wells to achieve a final concentration of 100 μM.Twenty-five μL of media were sampled from the wells 30. minutes afterthe addition of arachidonic acid for PGE₂ determination.

[0142] Cell viability—Cell viability was assessed by a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-basedcolorimetric assay (Sigma, St. Louis, Mo.). The MTT solution was addeddirectly to the wells after sampling for PGE₂ determination. Theabsorbance of each well was read at 580 nm using an ELISA plate reader.No toxicity was observed at the highest concentrations tested for any ofthe compounds.

[0143] Calculations—The median inhibitory concentration (IC₅₀) for PGE₂synthesis was calculated using CalcuSyn (BIOSOFT, Ferguson, Mo.). Thisstatistical package performs multiple drug dose-effect calculationsusing the median effect methods described by T-C Chou and P. Talaly[(1984) Quantitative analysis of dose-effect relationships: the combinedeffects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22,27-55.] hereby incorporated by reference.

[0144] Briefly, the analysis correlates the “Dose” and the “Effect” inthe simplest possible form: fa/fu=(C/C_(m))^(m), where C is theconcentration or dose of the compound and Cm is the median-effectivedose signifying the potency. Cm is determined from the x-intercept ofthe median-effect plot. The fraction affected by the concentration ofthe test material is fa and the fraction unaffected by the concentrationis fu (fu=1−fa). The exponent m is the parameter signifying thesigmoidicity or shape of the dose-effect curve. It is estimated by theslope of the median-effect plot.

[0145] The median-effect plot is a graph of x=log(C) vs y=log(fa/fu) andis based on the logarithmic form of Chou's median-effect equation. Thegoodness of fit for the data to the median-effect equation isrepresented by the linear correlation coefficient r of the median-effectplot. Usually, the experimental data from enzyme or receptor systemshave an r>0.96, from tissue culture an r>0.90 and from animal systems anr>0.85. In the cell-based studies reported here, all linear correlationcoefficients were greater than 0.90. Experiments were repeated threetimes on three different dates. The percent inhibition at each dose wasaveraged over the three independent experiments and used to calculatethe median inhibitory concentrations reported.

[0146] Results—The highly specific COX-2 inhibitor diisofluorophosphateexhibited a median inhibitory concentration in A549 cells of 1.19 μg/mLand did not inhibit PGE₂ synthesis in AGS cells at the highestconcentration tested of 25 μg/mL (Table 3). Rofecoxib, and celexocib,selective COX-2 drugs, were 27-, and 14-times, respectively, more potentinhibitors of PGE₂ synthesis in the target A549 cells than in thenon-target AGS gastric mucosal cells. This finding demonstrates not onlyCOX-2 selectivity, but also target-tissue selectivity consistent withtheir low gastrointestinal toxicity. Nimensulide, another new, selectiveCOX-2 inhibitor was equally as potent in the inhibition of PGE₂synthesis in both cell lines. The anti-inflammatory agent acetaminophen,purported to inhibit an unidentified isozyme of COX (COX-3) and havinglow gastrointestinal toxicity, inhibited PGE₂ biosynthesis in A549 cellsbut had no effect on PGE₂ synthesis in AGS gastric mucosal cells.

[0147] Alternatively and consistent with their demonstrated clinicalgastric toxicity, ibuprofen, aspirin and indomethacin all exhibited moreinhibition of PGE₂ synthesis in the AGS cell line than in the targetA549 cells. Salicylic acid, an anti-inflammatory agent that inhibits theexpression of COX-2 with little gastric irritation, was inactive in bothcell models. TABLE 3 Median inhibitory concentrations for test compoundsin the A549 and AGS cell lines. IC₅₀ A549 IC₅₀ AGS Compound [μg/mL][μg/mL] IC₅₀ AGS/IC₅₀ A549 Diisofluorophosphate 1.19 >25 >21 Rofecoxib0.081 2.21 27.3 Celexocib 0.004 0.055 13.8 Nimensulide 0.10 0.11 1.0Ibuprofen 0.10 0.05 0.50 Aspirin 0.48 0.09 0.19 Indomethacin 0.033 0.0020.002 Salicylic acid >25 >25 >1 Acetaminophen 0.607 >25 >41

[0148] These results validate the use of the AGS gastric mucosal cellline to evaluate potential gastrointestinal toxicity ofanti-inflammatory agents capable of inhibiting the synthesis of PGE₂.They also demonstrate cellular specificity in the action ofCOX-inhibiting compounds. A ratio of 1 for IC₅₀ AGS/IC₅₀ A549 indicatesIC₅₀'s that are the same for both the AGS cell and A549 cells. If theratio is higher than 1 for IC₅₀ AGS/IC₅₀ A549, then the inhibition ofPGE₂ is lower for the AGS cells. A lower inhibition of PGE₂ in AGS cellsis favorable because AGS cell line expresses more COX-1, which maintainsmucosal homeostasis.

EXAMPLE 3 Inhibition of PGE₂ Synthesis in Stimulated and NonstimulatedMurine Macrophages by Hops (Humulus lupulus) Compounds and Deriviatives

[0149] Summary—This example illustrates the potency of hops fractionsand derivatives to inhibit COX-2 synthesis of PGE₂ preferentially overCOX-1 synthesis of PGE₂ in the murine macrophage model.

[0150] Chemicals and reagents—Bacterial lipopolysaccharide (LPS; B E.coli 055:B5) was from Sigma (St. Louis, Mo.). Hops fractions (1) alphahop (1% alpha acids; AA), (2) aromahop OE (10% beta acids and 2%isomerized alpha acids, (3) isohop (isomerized alpha acids; IAA), (4)beta acid solution (beta acids BA), (5) hexahop gold (hexahydroisomerized alpha acids; HHIAA), (6) redihop (reduced isomerized-alphaacids; RIAA), (7) tetrahop (tetrahydro-iso-alpha acids THIAA) and (8)spent hops were obtained from Betatech Hops Products (Washington, D.C.,U.S.A.). The spent hops were extracted two times with equal volumes ofabsolute ethanol. The ethanol was removed by heating at 40° C. until aonly thick brown residue remained. This residue was dissolved in DMSOfor testing in RAW 264.7 cells. Unless otherwise noted, all standardreagents were obtained from Sigma (St. Louis, MO) and were the purestcommercially available. All other chemicals and equipment were asdescribed in Examples 1 and 2.

[0151] Cell culture—RAW 264.7 cells, obtained from American Type CultureCollection (Catalog #TIB-71, Manassas, Va.), were grown in Dulbecco'sModification of Eagle's Medium (DMEM, Mediatech, Herndon, Va.) andmaintained in log phase. The DMEM growth medium was made by adding 50 mLof heat inactivated FBS and 5 mL of penicillin/streptomycin to a 500 mLbottle of DMEM and storing at 4° C. The growth medium was warmed to 37°C. in water bath before use.

[0152] On day one of the experiment, the log phase RAW 264.7 cells wereplated at 8×10⁴ cells per well in 0.2 mL growth medium per well in a96-well tissue culture plate in the morning. At the end of the day one(6 to 8 h post plating), 100 μL of growth medium from each well wereremoved and replaced with 100 μL fresh medium.

[0153] A 1.0 mg/mL stock solution of LPS, used to induce the expressionof COX-2 in the RAW 264.7 cells, was prepared by dissolving 1.0 mg ofLPS in 1 mL DMSO. It was vortexed until dissolved and stored at 4° C.Before use, it was melted at room temperature or in a 37° C. water bath.

[0154] On day two of the experiment, test materials were prepared as1000X stock in DMSO. In 1.7 mL microfuge tubes, 1 mL DMEM without FBSwas added for test concentrations of 0.05, 0.10, 0.5, and 1.0 μg/mL. TwoμL of the 1000X DMSO stock of the test material was added to the 1 mL ofmedium without FBS. The tube contained the final concentration of thetest material concentrated 2-fold and placed tube in an incubator for 10minutes to equilibrate to 37° C.

[0155] For COX-2 associated PGE₂ synthesis, 100 μL of medium wereremoved from each well of the cell plates prepared on day one andreplaced with 100 μL of equilibrated 2X final concentration of the testcompounds. Cells were then incubated for 90 minutes. Twenty μL of LPSwere added to each well of cells to be stimulated to achieve a finalconcentration of 1 μg LPS/mL and the cells were incubated for 4 h. Thecells were further incubated with 5 μM arachidonic acid for 15 minutes.Twenty-five μL of supernatant medium from each well was transferred to aclean microfuge tube for the determination of PGE₂ released into themedium.

[0156] Following the LPS stimulation, the appearance of the cells wasobserved and viability was determined as described in Example 2. Notoxicity was observed at the highest concentrations tested for any ofthe compounds. Twenty-five μL of supernatant medium from each well wastransferred to a clean microfuge tube for the determination of PGE₂released into the medium. PGE₂ was determined and reported as previouslydescribed in Example 1.

[0157] For COX-1 associated PGE₂ synthesis, 100 μL of medium wereremoved from each well of the cell plates prepared on day one andreplaced with 100 μL of equilibrated 2X final concentration of the testcompounds. Cells were then incubated for 90 minutes. Next, instead ofLPS stimulation, the cells were incubated with 100 μM arachidonic acidfor 15 minutes. Twenty-five μL of supernatant medium from each well wastransferred to a clean microfuge tube for the determination of PGE₂released into the medium. The appearance of the cells was observed andviability was determined as described in Example 2. No toxicity wasobserved at the highest concentrations tested for any of the compounds.Twenty-five μL of supernatant medium from each well was transferred to aclean microfuge tube for the determination of PGE₂ released into themedium. PGE₂ was determined and reported as previously described inExample 1. The median inhibitory concentrations (IC₅₀) for PGE₂synthesis from both COX-2 and COX-1 were calculated as described inExample 2. TABLE 4 COX-2 and COX-1 inhibition in RAW 264.7 cells by hopfractions and derviatives COX-2 COX-1 IC₅₀ IC₅₀ Test Material [μg/mL][μg/mL] COX-1/COX-2 Alpha hop (AA) 0.21 6.2 30 Aromahop OE 1.6 4.1 2.6Isohop (IAA) 0.13 18 144 Beta acids (BA) 0.54 29 54 Hexahop (HHIAA) 0.293.0 11 Redihop (RIAA) 0.34 29 87 Tetrahop (THIAA) 0.20 4.0 21 Spent hops(EtOH) 0.88 21 24

[0158] As seen in Table 4, all hops fractions and derivative selectivelyinhibited COX-2 over COX-1 in this target macrophage model. This was anovel and unexpected finding. The extent of COX-2 selectivity for thehops derivatives IAA and RIAA, respectively, 144- and 87-fold, wasunanticipated. Such high COX-2 selectivity combined with low medianinhibitory concentrations, has not been previously reported for naturalproducts from other sources.

EXAMPLE 4 Hops Compounds and Derivatives Are Not Direct CyclooxygenaseEnzyme Inhibitors

[0159] Summary—This example illustrates that hops compounds andderivatives do not inhibit PGE₂ synthesis in A549 pulmonary epithelialcells at physiologically relevant concentrations when tested using theWHMA-COX-2 protocol.

[0160] Chemicals—Hops and hops derivatives used in this example werepreviously described in Example 3. All other chemicals were obtainedfrom suppliers as described in Examples 1 and 2.

[0161] Cells—A549 (human pulmonary epithelial) Cells were obtained fromthe American Type Culture Collection (Manassas, Va.) and sub-culturedaccording to the instructions of the supplier. The cells were routinelycultured at 37° C. with 5% CO₂ in RPMI 1640 containing 10% FBS, with 50units penicillin/mL, 50 μg streptomycin/mL, 5% sodium pyruvate, and 5%L-glutamine. On the day of the experiments, exponentially growing cellswere harvested and washed with serum-free RPMI 1640.

[0162] Log phase A549 cells were plated at 8×10⁴ cells per well with 0.2mL growth medium per well in a 96-well tissue culture plate. For thedetermination of PGE₂ inhibition by the test compounds, the procedure ofWarner et al. [(1999) Nonsteroid drug selectivities forcyclo-oxygenase-1 rather than cyclo- oxygenase-2 are associated withhuman gastrointestinal toxicity: a full in vitro analysis. Proc NatlAcad Sci U S A 96, 7563-7568], also known as the WHMA-COX-2 protocol wasfollowed with no modification. Briefly, 24 hours after plating of theA549 cells, interleukin-1β (10 ng/mL) was added to induce the expressionof COX-2. After 24 hr, the cells were washed with serum-free RPMI 1640and the test materials, dissolved in DMSO and serum-free RPMI, wereadded to the wells to achieve final concentrations of 25, 5.0, 0.5 and0.05 μg/mL. Each concentration was run in duplicate. DMSO was added tothe control wells in an equal volume to that contained in the testwells. Sixty minutes later, A23187 (50 μM) was added to the wells torelease arachidonic acid. Twenty-five μL of media were sampled from thewells 30 minutes later for PGE₂ determination.

[0163] Cell viability was assessed as previously described in Example 2.No toxicity was observed at the highest concentrations tested for any ofthe compounds. PGE₂ in the supernatant medium was determined andreported as previously described in Example 1.

[0164] The median inhibitory concentration (IC₅₀) for PGE₂ synthesis wascalculated as previously described in Example 2.

[0165] Results—At the doses tested, the experimental protocol failed tocapture a median effective concentration of any of the hops extracts orderivatives. Since the protocol requires the stimulation of COX-2expression prior to the addition of the test compounds, the likelyanswer to the failure of the test materials to inhibit PGE₂ synthesis isthat their mechanism of action is to inhibit the expression of the COX-2isozyme and not activity directly. While some direct inhibition can beobserved using the WHMA-COX-2 protocol, this procedure is inappropriatein evaluating the anti-inflammatory properties of hops compounds orderivatives of hops compounds.

EXAMPLE 5 Lack of Inhibition of PGE₂ Synthesis in Gastric Mucosal Cellsby Hops (Humulus lupulus) Compounds and Deriviatives

[0166] Summary—This example illustrates the lack of PGE₂ inhibition byhops fractions and in the AGS human gastric mucosal cell line implyinglow gastric irritancy potential of these compounds.

[0167] Chemicals and reagents were used as described in Example 3. AGScells were grown and used for testing hops compounds and derivatives asdescribed in Example 2. PGE₂ was determined and reported as previouslydescribed in Example 1. The median inhibitory concentrations (IC₅₀) forPGE₂ synthesis from AGS cells were calculated as described in Example 2.TABLE 5 Lack of PGE₂ inhibition in AGS gastric mucosal cells by hopfractions and derivatives IC₅₀ AGS Test Material [μg/mL] Alpha hop(AA) >25 Aromahop OE >25 Isohop (IAA) >25 Beta acids (BA) >25 Hexahop(HHIAA) >25 Redihop (RIAA) >25 Tetrahop (THIAA) >25 Spent hops (EtOH)>25

[0168] As seen in Table 5, all hops fractions and Derivatives wereunable to inhibit PGE₂ synthesis by 50% or more at the highestconcentrations tested in the AGS gastric mucosal cell line. Based on theanti-inflammatory potency exhibited by these fractions in targetmacrophages, this was a novel and unexpected finding.

EXAMPLE 6 Normalization of Joint Function following Trauma

[0169] A representative composition of the preferred embodiments as adietary supplement would be in an oral formulation, i.e. tablets or gelcaps that would supply one of the following combinations: 0.1 to 10 mgisocohumulone/kg per day; 0.01 to 10 mg dihydro-adhumulone/kg per day;0.01 to 10 mg tetrahydro-isocohumulone/kg per day; 0.01 to 10 mg/kg perday of hexahydro-isohumulone/kg per day for a 70 kg person.

[0170] Normalization of joint movement following physical trauma due toexercise or repetitive movement stress would be expected to occurfollowing two to ten doses. This result would be expected in allanimals.

EXAMPLE 7 Clinical Effectiveness of Lotion Formulations in the Treatmentof Acne Rosacea

[0171] A lotion designed to contain one of the following:

[0172] 1. 0.1% wt of the isomerized alpha-acid isocohumulone;

[0173] 2. 0.1% wt of the reduced isomerized alpha-aciddihydro-adhumulone;

[0174] 3. 0.1% wt of the tetrahydroisoalpha-acidtetrahydro-isocohumulone; or

[0175] 4. 0.1% wt hexahydro-isohumulone

[0176] is applied to affected areas of patients who have exhibited acnerosacea as diagnosed by their health practitioner and confirmed by anindependent board-certified dermatologist.

[0177] Self-evaluation tests and are administered one week prior to thestudy to quantify the surface area affected and redness. In addition,similar variables are scored by the professional clinical staff notaware of the patients treatment status. These evaluations are repeatedon Days 0, 7, 14 and 21.

[0178] Patients are randomly assigned to the test formulation or placeboat the start of the study. The test formulation and placebo are appliedto the affected area one or two times per day. Treatment for healthconditions such as diabetes, hypertension, etc. is allowed during thestudy. Scores are statistically compared between the test formulationand the placebo for each of the four observational periods. Patientstreated with the composition of the preferred embodiments in a lotionformulation are considered improved if the patients' scores improve bygreater than 20% from the pre-test scores within each categoryevaluated. The percentage of persons exhibiting improvement is comparedbetween the combination formulations and the placebo control. Thedifference between the two groups is considered statisticallysignificant if the probability of rejecting the null hypothesis whentrue is less than five percent.

EXAMPLE 8 Clinical Effectiveness of a Lotion Formulation in theTreatment of Psoriasis

[0179] This example is performed in the same manner as described inExample 7, except that the composition is applied to affected areas ofpatients who have exhibited psoriasis as diagnosed by their ownpractitioner and confirmed by an independent board-certifieddermatologist. Self-evaluation tests are administered one week prior tothe study to quantify the surface area affected and skin condition. Inaddition, similar variables are scored by the professional clinicalstaff not aware of the patients treatment status. These evaluations arerepeated on Days 0, 7, 30 and 60.

[0180] Patients are randomly assigned to the test formulation or placeboat the start of the study. The test formulation and placebo are appliedto the affected area one or two times per day. Treatment for healthconditions such as diabetes, hypertension, etc. is allowed during thestudy. Scores are statistically compared between the test formulationand the placebo for each of the four observational periods. Patientstreated with the composition of the preferred embodiments as the testlotion formulation are considered improved if the patients' scoresimprove by greater than 20% from the pre-test scores within eachcategory evaluated. The percentage of persons exhibiting improvement iscompared between the test formulation and the placebo control. Thedifference between the two groups is considered statisticallysignificant if the probability of rejecting the null hypothesis whentrue is less than five percent.

EXAMPLE 9 Clinical Effectiveness of a Formulation in the treatment ofAlzheimer's Disease

[0181] An oral formulation as described in Example 6 is administered topatients who have manifested an early stage of Alzheimer's Disease (AD),as diagnosed by their practitioner and confirmed by an independentboard-certified neurologist. Two weeks before the clinical trial, thepatients undergo appropriate psychoneurological tests such as the MiniMental Status Exam (MMSE), the Alzheimer Disease Assessment Scale(ADAS), the Boston Naming Test (BNT), and the Token Test (TT).Neuropsychological tests are repeated on Day 0, 6 weeks and 3 months ofthe clinical trial. The tests are performed by neuropsychologists whoare not aware of the patient's treatment regimen.

[0182] Patients are randomly assigned to the test formulation or placeboat the start of the study. The test formulation and placebo are takenorally one or two times per day. Treatment for conditions such asdiabetes, hypertension, etc. is allowed during the study. Scores arestatistically compared between the test formulation and the placebo foreach of the three observational periods. Without treatment, the naturalcourse of AD is significant deterioration in the test scores during thecourse of the clinical trial. Patients treated with the composition ofthe preferred embodiments as the test formulation are consideredimproved if the patients' scores remain the same or improve during thecourse of the clinical trial.

EXAMPLE 10 Oral Formulation in the Treatment and Prevention of ColonCancer

[0183] An oral formulation as described in Example 6 is administered topatients who have manifested an early stage of colon cancer as diagnosedby their own practitioner and confirmed by a independent board-certifiedoncologist.

[0184] Patients are randomly assigned to the test formulation or aplacebo at the start of the study. The test formulation and placebo aretaken orally one or two times per day. Treatment for conditions such asdiabetes, hypertension, etc. is allowed during the study. Endoscopicevaluations are made at one, two, six and twelve months. Evidence ofreappearance of the tumor during any one of the four follow-up clinicalvisits is considered a treatment failure. The percentage of treatmentfailures is compared between the test formulation and the placebocontrol. Under the experimental conditions described, the test materialis expected to decrease the tumor incidence with respect to the controlgroup. The difference between the two groups is considered statisticallysignificant if the probability of rejecting the null hypothesis whentrue is less than five percent.

EXAMPLE 11 Oral Formulation of the Treatment of Irritable Bowel Syndrome

[0185] An oral formulation as described in Example 6 is administered topatients who have manifested irritable bowel syndrome as diagnosed bytheir practitioner. Normal bowel functioning is restored within 48hours.

EXAMPLE 12 Normalization of Joint Functioning in Osteoarthritis

[0186] Using compositions described in Example 6 normalization of jointstiffness due to osteoarthritis occurs following five to twenty doses,in the presence or absence of glucosamine or chondroitin sulfate. Inaddition, the composition does not interfere with the normal jointrebuilding effects of these two proteoglycan constituents, unliketraditional non-steroidal anti-inflammatory agents.

EXAMPLE 13 Mite Dust Allergens Activate PGE₂ Biosynthesis in A549Pulmonary Cells

[0187] Summary—This example illustrates that house mite dust allergenscan induce PGE₂ biosynthesis in pulmonary epithelial cells.

[0188] Background

[0189] Sensitivity to allergens is a problem for an increasing number ofconsumers. This issue has been complicated by a surprising increase inasthma over the past few years. Asthma suffers are especially sensitiveto airborne allergens. Allergy rates are also on the rise. This givesrise to increased awareness of the causes of allergy symptoms and how todecrease the associated discomfort. Approximately 10% of the populationbecome hypersensitized (allergic) upon exposure to antigens from avariety of environmental sources. Those antigens that induce immediateand/or delayed types of hypersensitivity are known as allergens. Theseinclude products of grasses, trees, weeds, animal dander, insects, food,drugs, and chemicals. Genetic predisposition of an individual isbelieved to play a role in the development of immediate allergicresponses such as atopy and anaphylaxis whose symptoms include hayfever, asthma, and hives.

[0190] Many allergens are protein-based molecules, and these proteinallergens can originate from many sources. It has been known for sometime that one of the most common sources of allergens in a house is fromdust mites. Of course, as is the case with all allergens, only certainpeople are allergic to dust mite allergens. But this group of people canbe quite large in many areas, especially in hot humid areas. Forexample, in the southeastern United States of America, where it is bothhot and humid for much of the year, the incidence of house dust miteallergies in the general population can be as high as 25%. House dustmites thrive in plush carpets, overstuffed upholstery, cushy bedcomforters and the like.

[0191] Methods

[0192] Mite dust allergen isolation—Dermatophagoides farinae are theAmerican house dust mite. D. farinae were cultured on a 1:1 ratio ofPurina Laboratory Chow (Ralston Purina, Co, St. Louis, Mo.) andFleischmann's granulated dry yeast (Standard Brands, Inc. New York,N.Y.) at room temperature and 75% humidity. Live mites were aspiratedfrom the culture container as they migrated from the medium, killed byfreezing, desiccated and stored at 0% humidity. The allergenic componentof the mite dust was extracted with water at ambient temperature.Five-hundred mg of mite powder were added to 5 mL of water (1:10 w/v) ina 15 mL conical centrifuge tube (VWR, Rochester, N.Y.), shaken for oneminute and allowed to stand overnight at ambient temperature. The nextday, the aqueous phase was filtered using a 0.2 μm disposable syringefilter (Nalgene, Rochester, N.Y.). The filtrate was termed mite dustallergen and used to test for induction of PGE₂ biosynthesis in A549pulmonary epithelial cells.

[0193] Cell culture and treatment—This experiment involved the humanairway epithelial cell line, A549 (American Type Culture Collection,Bethesda, Md.). The cells were cultured and treated as previouslydescribed in Example 2. Mite allergen was added to the culture medium toachieve a final concentration of 1000 ng/mL. Twenty-four hours later,the culture medium was sampled for PGE₂ concentration.

[0194] PGE₂ assay—Determination of PGE₂ in the culture medium wasperformed as previously described in Example 1.

[0195] Statistical analysis—Means of eight replicates per treatment werecomputed using Excel® spreadsheets (Microsoft, Redmond, Wash.).

[0196] Results

[0197] Mite allergen treatment increased PGE₂ biosynthesis 6-fold inA549 cells relative to the solvent treated controls (FIG. 5).

EXAMPLE 14 Hops Derivatives Inhibit Mite Dust Allergen Activation ofPGE₂ Biosynthesis in A549 Pulmonary Cells

[0198] Summary—This example illustrates that hops derivatives arecapable of inhibiting the PGE₂ stimulatory effects of mite dustallergens in A549 pulmonary cells.

[0199] Methods

[0200] The cell line and testing procedures are as described in Example14. In addition to mite dust allergen, test materials included Hopsfractions (1) alpha hop (1% alpha acids; AA), (2) aromahop OE (10% betaacids and 2% isomerized alpha acids , (3) isohop (isomerized alphaacids; IAA), (4) beta acid solution (beta acids BA), (5) hexahop gold(hexahydro isomerized alpha acids; HHIAA), (6) redihop (reducedisomerized-alpha acids; RIAA), and (7) tetrahop (tetrahydro-iso-alphaacids THIAA). Test materials at a final concentration of 10 μg/mL wereadded 60 minutes prior to the addition of the mite dust allergen.

[0201] Results

[0202] Table 7 depicts the extent of inhibition of PGE₂ biosynthesis byhops derivatives in A549 pulmonary cells stimulated by mite dustallergen. All hops derivatives were capable of significantly inhibitingthe stimulatory effects of mite dust allergens. TABLE 7 PGE₂ inhibitionby hops derviatives in A549 pulmonary epithelial cells stimulated bymite dust allergen Percent Inhibition of Test Material PGE₂ BiosynthesisAlpha hop (AA) 81 Aromahop OE 84 Isohop (IAA) 78 Beta acids (BA) 83Hexahop (HHIAA) 82 Redihop (RIAA) 81 Tetrahop (THIAA) 76

[0203] In conclusion, it would be useful to identify a naturalformulation of compounds that would inhibit expression of COX-2, inhibitprostaglandin synthesis selectively in target cells, or inhibitinflammation response selectively in target cells.

[0204] A preferred embodiment comprises compositions containing at leastone fraction isolated or derived from hops (Humulus lupulus). Examplesof fractions isolated or derived from hops are alpha acids, isoalphaacids, reduced isoalpha acids, tetra-hydroisoalpha acids,hexa-hydroisoalpha acids, beta acids, and spent hops. Preferredcompounds of fractions isolated or derived from hops, include, but arenot limited to, humulone, cohumulone, adhumulone, isohumulone,isocohumulone, isoadhumulone, dihydro-isohumulone,dihydro-isocohumulone, dihydro-adhumulone, tetrahydro-isohumulone,tetrahydro-isocohumulone, tetrahydro-adhumulone, hexahydro-isohumulone,hexahydro-isocohumulone, and hexahydro-adhumulone. Preferred compoundscan also bear substituents, such as halogens, ethers, and esters.

[0205] It will be readily apparent to those skilled in the art thatvarious changes and modifications of an obvious nature may be madewithout departing from the spirit of the invention, and all such changesand modifications are considered to fall within the scope of theinvention as defined by the appended claims. Such changes andmodifications would include, but not be limited to, the incipientingredients added to affect the capsule, tablet, lotion, food or barmanufacturing process as well as vitamins, herbs, flavorings andcarriers. Other such changes or modifications would include the use ofother herbs or botanical products containing the combinations of thepreferred embodiments disclosed above. Many additional modifications andvariations of the embodiments described herein may be made withoutdeparting from the scope, as is apparent to those skilled in the art.The specific embodiments described herein are offered by way of exampleonly.

What is claimed is:
 1. A method of modulating the inflammatory responsein cells, the method comprising contacting the cells with a compositioncomprising a fraction isolated or derived from hops.
 2. A method oftreating or inhibiting a pathological condition in a mammal associatedwith tissue-specific activation of inflammation, the method comprisingadministering to the mammal a composition comprising a fraction derivedfrom hops.
 3. The method of claim 2, wherein the fraction derived fromhops is selected from the group consisting of isoalpha acids, reducedisoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids,beta acids, and spent hops.
 4. The method of claim 2, wherein thefraction derived from hops comprises a compound of a supragenus havingthe formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; wherein R″ is selected from the groupconsisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and wherein R,T, X, and Z are independently selected from the group consisting of H,F, Cl, Br, I, and π orbital, with the proviso that if one of R, T, X, orZ is a π orbital, then the adjacent R, T, X, or Z is also a π orbital,thereby forming a double bond.
 5. The method of claim 2, wherein thefraction derived from hops comprises a compound of Genus A having theformula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.
 6. Themethod of claim 2, wherein the fraction derived from hops comprises acompound of Genus B having the formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.
 7. Themethod of claim 2, wherein the fraction derived from hops comprises acompound selected from the group consisting of cohumulone, adhumulone,isohumulone, isocohumulone, isoadhumulone, dihydro-isohumulone,dihydro-isocohumulone, dihydro-adhumulone, tetrahydro-isohumulone,tetrahydro-isocohumulone, tetrahydro-adhumulone, hexahydro-isohumulone,hexahydro-isocohumulone, and hexahydro-adhumulone.
 8. The method ofclaim 2, wherein the composition comprises about 0.5 to 10000 mg of thefraction derived from hops.
 9. The method of claim 8, wherein thecomposition comprises about 50 to 7500 mg of the fraction derived fromhops.
 10. The method of claim 2, wherein the composition comprises about0.001 to 10 weight percent of the fraction derived from hops.
 11. Themethod of claim 10, wherein the composition comprises about 0.1 to 1weight percent of the fraction derived from hops.
 12. The method ofclaim 2, wherein the pathological condition is selected from the groupconsisting of autoimmune diseases, inflammatory diseases, neurologicaldiseases, and cancer.
 13. The method of claim 2, wherein thepathological condition is selected from the group consisting ofinflammation, inflammation-associated disorders, arthritis, asthma,bronchitis, menstrual cramps, tendonitis, bursitis, skin-relatedconditions, gastrointestinal conditions, cancer, ophthalmic diseases,pulmonary inflammation, nervous system disorders, allergic rhinitis,respiratory distress syndrome, endotoxin shock syndrome,atherosclerosis, and central nervous damage.
 14. The method of claim 2,wherein the composition further comprises a pharmaceutically acceptablecarrier.
 15. The method of claim 2, wherein the composition isadministered orally, topically, parenterally, or rectally.
 16. A methodof modulating the amount of cyclooxygenase-2 (COX-2) activity in targetcells without substantially modulating COX-2 activity in non-targetcells, the method comprising contacting the cells with a fractionderived from hops.
 17. The method of claim 16, wherein the non-targetcells are also contacted with said fraction derived from hops.
 18. Themethod of claim 16, wherein the contacting step is in vivo.
 19. Themethod of claim 16, wherein the COX-2 activity is modulated byinhibition of COX-2 gene.
 20. A method of treating or inhibiting apathological condition in a mammal involving inhibiting inducibility oractivity of cyclooxygenase-2 (COX-2), the method comprisingadministering to the mammal a composition comprising a fraction derivedfrom hops.
 21. The method of claim 20, wherein the fraction derived fromhops is selected from the group consisting of isoalpha acids, reducedisoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids,beta acids, and spent hops.
 22. The method of claim 20, wherein thefraction derived from hops comprises a compound of a supragenus havingthe formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; wherein R″ is selected from the groupconsisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and wherein R,T, X, and Z are independently selected from the group consisting of H,F, Cl, Br, I, and π orbital, with the proviso that if one of R, T, X, orZ is a π orbital, then the adjacent R, T, X, or Z is also a π orbital,thereby forming a double bond.
 23. The method of claim 20, wherein thefraction derived from hops comprises a compound of Genus A having theformula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.
 24. Themethod of claim 20, wherein the fraction derived from hops comprises acompound of Genus B having the formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.
 25. Themethod of claim 20, wherein the fraction derived from hops comprises acompound selected from the group consisting of cohumulone, adhumulone,isohumulone, isocohumulone, isoadhumulone, dihydro-isohumulone,dihydro-isocohumulone, dihydro-adhumulone, tetrahydro-isohumulone,tetrahydro-isocohumulone, tetrahydro-adhumulone, hexahydro-isohumulone,hexahydro-isocohumulone, and hexahydro-adhumulone.
 26. The method ofclaim 20, wherein the pathological condition is selected from the groupconsisting of wherein the pathological condition is selected from thegroup consisting of inflammation, inflammation-associated disorders,arthritis, asthma, bronchitis, menstrual cramps, tendonitis, bursitis,skin-related conditions, gastrointestinal conditions, cancer, ophthalmicdiseases, pulmonary inflammation, nervous system disorders, allergicrhinitis, respiratory distress syndrome, endotoxin shock syndrome,atherosclerosis, and central nervous damage.
 27. The method of claim 20,wherein the composition further comprises a pharmaceutically acceptablecarrier.
 28. The method of claim 20, wherein the composition isadministered orally, topically, parenterally, or rectally.
 29. A methodof inhibiting prostaglandin synthesis selectively in target cells, themethod comprising contacting the cells with a fraction derived fromhops.
 30. The method of claim 29, wherein the fraction derived from hopsis selected from the group consisting of-isoalpha acids, reducedisoalpha acids, tetra-hydroisoalpha acids, hexa-hydroisoalpha acids,beta acids, and spent hops.
 31. The method of claim 29, wherein thefraction derived from hops comprises a compound of a supragenus havingthe formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; wherein R″ is selected from the groupconsisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃; and wherein R,T, X, and Z are independently selected from the group consisting of H,F. Cl, Br, I, and π orbital, with the proviso that if one of R, T, X, orZ is a π orbital, then the adjacent R, T, X, or Z is also a π orbital,thereby forming a double bond.
 32. The method of claim 29, wherein thefraction derived from hops comprises a compound of Genus A having theformula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.
 33. Themethod of claim 29, wherein the fraction derived from hops comprises acompound of Genus B having the formula:

wherein R′ is selected from the group consisting of carbonyl, hydroxyl,OR, and OCOR, wherein R is alkyl; and wherein R″ is selected from thegroup consisting of CH(CH₃)₂, CH₂CH(CH₃)₂, and CH(CH₃)CH₂CH₃.
 34. Themethod of claim 29, wherein the fraction derived from hops comprises acompound selected from the group consisting of cohumulone, adhumulone,isohumulone, isocohumulone, isoadhumulone, dihydro-isohumulone,dihydro-isocohumulone, dihydro-adhumulone, tetrahydro-isohumulone,tetrahydro-isocohumulone, tetrahydro-adhumulone, hexahydro-isohumulone,hexahydro-isocohumulone, and hexahydro-adhumulone.